The amino acid p-benzoyl-L-phenylalanine, @-Bz)Phe, has been incorporated into substance P (SP), Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2, to localize the agonist-binding domains of the human neurokinin-1 (NK-1) receptor overexpressed in a transfected mammalian cell line. The NK-1 -specific agonist [Pro9]SP was modified at position 8 by @-Bz)Phe and acylated at the N-terminus by a biotinyl sulfone reporter via a 5-aminopentanoyl spacer. After photolysis, the biotinyl sulfone moiety allowed easy and efficient removal of biotinyiated fragments from the complex incubation mixture with streptavidin-coated beads. Direct elution from the beads with the matrix used for matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS), which was facilitated by saturation of streptavidin sites with biotin, and subsequent MALDI-TOF mass spectrometry analysis allowed identification of the NK-1 fragments obtained after photolysis and proteolytic digestion. Trypsin digestion and combined trypsinlstaphylococcus aureus V8 protease enzymatic cleavage established that the site of covalent attachment of the photolabelled SP resides in the second extracellular loop, Thr173 -Arg177. Cyanogen bromide cleavage shows that the probe is covalently attached to the methyl group of a methionine residue from human NK-1. These experiments identified Met174 as the modified residue.Keywords: tachykinin receptor; photolabelling ; substance P; Chinese hamster ovary cells ; mass spectrometry.Chimeric tachykinin NK-UNK-3 or NK-UNK-2 receptors and mutated neurokinin-1 (NK-1) receptors have been constructed to probe the binding domains of NK-1 agonists and antagonists [ 1 -151. Both extracellular domains (N-terminal residues and extracellular loops) and residues from the transmembrane segments [I, 3, 5, 7, 8, 12, 13, 151 have been identified as important determinants for the binding of substance P and specific NK-1 agonists. However, as is evident from the conflicting data, these results do not imply that these critical residues of the NK-1 receptor directly interact with the amino acids of substance P; these residues may only confer the proper folding to the binding site for the agonist in the protein [13, 151. Affinity labelling of the receptor using photoreactive ligands constitutes a complementary strategy to mutagenesis [16]. The use of photolabelled analogues of the ligand-incorporating reactive probes in various positions allows mapping of the agonistbinding site. Theoretically, this strategy seems more precise than mutagenesis for the determination of the amino acids that constitute the binding pocket in the protein. However, numerous photolabelling experiments should be performed to describe the foot-printing of the ligand bound to its receptor. It should be noted that (a) important residues of the ligand cannot usually be modified (loss of the binding potency); the residue that is easily replaced by a photoreactive probe may not contribute to the binding contacts with the protein; and (b) when a compromis...

Extraction of biotinylated peptides by streptavidin magnetic beads has been directly coupled to the MALDI-TOF mass analysis. The elution of peptides from the beads is achieved by first mixing the beads with the MALDI matrix solution and removing, after a few minutes, the beads with a magnet; then, the matrix solution containing the biotinylated peptide is directly mass analyzed by MALDI. Three examples are presented to show the capabilities of this procedure to detect biotinylated peptides present at very low concentrations in complex mixtures. Detection limits of less than 100 finol can be achieved. Such a coupling strategy is of great interest to investigate peptide/ protein interactions.

A new series of 4-anilinoquinolines with two proton-accepting side chains has been synthesized. Antimalarial activity and levels of cytotoxicity upon both MRC-5 cells and macrophages were found to be highly dependent upon the features of these side chains. Several compounds were found to be active in the low nanomolar range, against both chloroquine-sensitive and -resistant strains of Plasmodium falciparum in vitro. From among them, a morpholino derivative cured mice infected by Plasmodium berghei and displayed a lower toxicity than amodiaquine upon mouse macrophages.

Forty bis(9-amino-6-chloro-2-methoxyacridines), in which acridine moieties are joined by alkanediamines, polyamines, or polyamines substituted by a side chain, were synthesized and tested for their in vitro activity upon the erythrocytic stage of Plasmodium falciparum, trypomastigote stage of Trypanosoma brucei, and amastigote stage of Trypanosoma cruzi and Leishmania infantum as well as for their cytotoxic effects upon MRC-5 cells. Results clearly showed the importance of the nature of the linker and of its side chain for antiparasitic activity, cytotoxicity, and cellular localization. Among several compounds devoid of cytotoxic effects at 25 microM upon MRC-5 cells, one displayed IC(50) values ranging from 8 to 18 nM against different P. falciparum strains while three others totally inhibited T. brucei at 1.56 microM.

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Bisquinoline heteroalkanediamines were structurally modified in order to study the effects of enhanced bulkiness and rigidity on both their activity on strains of Plasmodium falciparum expressing different degrees of chloroquine (CQ) resistance and their cytotoxicity toward mammalian cells. While cyclization yielded molecules of greater rigidity that were not more active than their linear counterparts, they were characterized by an absence of cytotoxicity. Alternatively, dimerization of these compounds led to tetraquinolines that are very potent for CQ-resistant strains and noncytotoxic.

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